A novel method for quickly cloning genes with multiple DNA fragments———one step cloning technique using isoschizomer-heterotail restriction endonuclease (IHRE) is described. Up to six DNA segments are ligated by using only one restriction endonuclease in this method. Comparing with routine method，it is simple, fast, economical and generates products with higher purity and achievement. Light chain of human enterokinase, DNA multi-epitope vaccine to HSV2 have been designed and successfully constructed via this method.